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Shoot multiplication induced by exogenous cytokinins (CKs) has been commonly used in <i>Phalaenopsis</i> micropropagation for commercial production. Despite this, mechanisms of CKs action on shoot multiplication remain unclear in <i>Phalaenopsis</i>. In this study, we first identified key CKs metabolic genes, including six isopentenyltransferase (<i>PaIPTs</i>), six cytokinin riboside 5′ monophosphate phosphoribohydrolase (<i>PaLOGs</i>), and six cytokinin dehydrogenase (<i>PaCKXs</i>), from the <i>Phalaenopsis</i> genome. Then, we investigated expression profiles of these CKs metabolic genes and endogenous CKs dynamics in shoot proliferation by thidiazuron (TDZ) treatments (an artificial plant growth regulator with strong cytokinin-like activity). Our data showed that these CKs metabolic genes have organ-specific expression patterns. The shoot proliferation in vitro was effectively promoted with increased TDZ concentrations. Following TDZ treatments, the highly expressed CKs metabolic genes in micropropagated shoots were <i>PaIPT1</i>, <i>PaLOG2,</i> and <i>PaCKX4</i>. By 30 days of culture, TDZ treatments significantly induced CK-ribosides levels in micropropagated shoots, such as <i>t</i>ZR and iPR (2000-fold and 200-fold, respectively) as compared to the controls, whereas <i>c</i>ZR showed only a 10-fold increase. Overexpression of <i>PaIPT1</i> and <i>PaLOG2</i> by agroinfiltration assays resulted in increased CK-ribosides levels in tobacco leaves, while overexpression of <i>PaCKX4</i> resulted in decreased CK-ribosides levels. These findings suggest de novo biosynthesis of CKs induced by TDZ, primarily in elevation of <i>t</i>ZR and iPR levels. Our results provide a better understanding of CKs metabolism in <i>Phalaenopsis</i> micropropagation.